The polycystic ovary syndrome (PCOS) affects ~7% of women and ~70% demonstrate insulin resistance, with the resulting hyperinsulinemia stimulating androgen excess. The economic burden of the disorder is estimated to exceed 4 billion dollars annually, in the U.S. and during the reproductive life span alone. The broad long-term objective of our studies is to establish the molecular etiology(s) of the PCOS-associated insulin resistance. Overall, little is know about the molecular aspects of insulin signaling in PCOS. Insulin-stimulated glucose uptake is deficient in PCOS, suggesting an alteration along the IRS/PI-3 kinase/Akt cascade, although the mitogenic activity and MAPK pathway appears unaffected. Insulin receptor (IR) tyrosine autophosphorylation also appears to be lower, and serine phosphorylation higher. In addition, we have obtained preliminary data indicating that PCOS adipocytes have deficient serine (inhibitory) and increased tyrosine (activating) glycogen synthase kinase-3 (GSK3) phosphorylation, consistent with enhanced GSK3 action. This data suggests that GSK3 dysregulation may represent a novel mechanism for insulin resistance in PCOS. We propose the following studies: Aim 1: To determine the role that defective regulation of GSK3 plays in mediating the abnormal IR signaling and glucose transport of PCOS;we will phenotype, including performing a frequently sampled intravenous glucose tolerance test, 70 PCOS patients and 70 matched controls;and in the adipocytes of these subjects determine the association of GSK3 activity with 2-deoxyglucose uptake;the content of regulators and substrates for GSK phosphorylation, determined by RT-PCR and/or Western blot (including total and phosphorylated IR substrate-1 and 2 [IRS-1/2], Akt, the PI-3 kinase subunits p110a and p110p, the 220-kDa A-kinase anchoring protein [AKAP220], protein kinase C [PKC], p70S6K, and the 2 GSK3-binding proteins known as FRAT1 and FRAT2);the impact of specific PKA, PKB (Akt), and PKC inhibition;in PCOS, the impact of specific GSK3 inhibition;and, in controls, the effect of GSKSbeta upregulation, using adenoviral-mediated transfection. Aim 2: To test whether abnormal signaling of the IRS/PI-3 kinase/Akt, but not the MAPK cascade, is present in PCOS;determining the degree of IR binding and 2-deoxyglucose uptake;and by RT-PCR and/or Western blot, the total content and phosphorylation in response to insulin of the IR, total and translocated GLUT-4, and of critical intermediate proteins (e.g. FKHR of the PI-3 kinase/Akt cascade: c-Raf, MEK-1, ERK1/2, pQORSK, and the translational regulator p70S6, of the ERK1/2 cascade: JNK of the SAPK/JNK cascade;and p38 MAPK of the P38MAPK cascade). Overall, these studies have the potential of elucidating the etioloaic mechanism(s) in PCOS, and guiding our search for therapies and molecular markers.